GLIPR2 Promotes Migration,invasion And EMT Progression Of NSCLC By Activating STAT3 Signaling Pathway Mediated By CAV1

Objective: In view of the extremely high incidence and mortality of lung cancer as a tumor killer in China,there is an urgent need to find new molecular markers to improve the situation.GLIPR2,also known as GAPR-1 or C9orf19 with a molecular weight of 17 k Da,is a member of the CAP superfamily.It is a peripheral membrane binding protein mainly located in the lipid-rich microdomain of the Golgi membrane.Immunohistochemistry suggested that GLIPR2 was associated with poor prognosis in lung cancer patients.GLIPR2’s involvement in the active regulation of EMT has been reported,and it is believed that EMT is a major contributor to its development.EMT is thought to be a key factor in the development of metastasis,relapse and drug resistance.However,there is no evidence that GLIPR2 plays a role in regulating EMT.Therefore,it is important to study the relationship between EMT and GLIPR2.Through GSEA gene enrichment analysis and STRING biogenic database analysis,a powerful relationship between GLIPR2 and STAT3 signaling was discovered.STAT3 is a critical nuclear transcription factor that facilitates the transmission of extracellular signals from the cell surface to the nucleus and the activation of gene transcription.STAT3 is abnormally activated and is involved in various cancer progression processes.This makes us wonder whether GLIPR2 modulates STAT3 signaling to control the malignant progression of NSCLC.Through biogenetic analysis and literature reports,we found a binding protein CAV1 that can interact with GLIPR2,so we constructed GLIPR2 mutant plasmid to verify our conjecture.To sum up,these findings shed light on the particular way GLIPR2 plays a role in the progression of lung cancer,thus offering a fresh foundation for GLIPR2 to become a potential target for clinical treatment of lung cancer.Methods:1.Immunohistochemical analysis of 118 NSCLC patients revealed the expression of GLIPR2 protein,and a correlation between GLIPR2 and clinicopathologic factors such as sex,age,lymph node metastasis,clinical stage,and tumor differentiation level.2.Next,we will construct plasmids for GLIPR2 overexpression and interference and investigate the mechanism of GLIPR2 affecting cell biology and specific related mechanisms.3.Expression of migration and invasion-associated proteins and EMT-associated proteins was examined by Western Blot after GLIPR2 overexpression and knockdown.4.To identify GLIPR2’s ability to function and verify it with the pathway inhibitor,GSEA seeks to examine relevant signaling pathways.5.GLIPR2 interacting proteins were analyzed and predicted by STRING protein interaction database,GEPIA database and Uni Prot database,and immunofluorescence co-localization and immunoco-precipitation experiments were conducted to explore GLIPR2 interacting proteins.6.Constructing the GLIPR2 splicosome plasmid,Western Blot,Transwell invasion,and scratch migration assays were conducted to investigate the specific GLIPR2 domain binding to the acting protein and whether it performs biological functions throughout this domain.Results:1.Immunohistochemistry suggested that GLIPR2 was strongly associated with poor prognosis in lung cancer patients.2.Cytofunctional experiments revealed that GLIPR2 overexpression stimulated NSCLC cell migration and infiltration capacity and EMT progression,and the opposite results were obtained after Gl IPR2 downregulation.3.Gene Set Enrichment Analysis(GSEA)and transcriptome sequencing indicated that GLIPR2 was closely related to the STAT3 signaling pathway,which was confirmed using the STAT3 signaling inhibitor.GLIPR2 may promote NSCLC migration,invasion,and EMT by regulating the STAT3 signaling pathway.4.The direct interaction between GLIPR2 and CAV1 was confirmed by STRING protein interaction database,GEPIA database and Uni Prot database,immunofluorescence colocalization and immunocoprecipitation experiments.5.Western Blot,transwell,and cell scratch demonstrated that GLIPR2 can positively regulate STAT3 pathway by binding with CAV1 to promote tumor cell migration,invasion,and EMT progression.Conclusion:1.A positive correlation exists between lymph node metastasis,tumor differentiation,and a high TNM stage in non-small cell lung cancer,which is accompanied by a high expression of GLIPR2.2.STAT3 signaling pathway activation by GLIPR2 can foster tumor cell migration,invasion,and EMT progression.3.GLIPR2 can bind directly to CAV1.4.GLIPR2 can activate the STAT3 signaling pathway by binding with CAV1 to promote tumor cell migration,invasion,and malignant progression of EMT.